ADDIS ABABA UNIVERSITY INSTITUTE OF BIOTECHNOLOGY Genetic Diversity and Population Structure of Zymoseptoria tritici Populations of Southern Ethiopia Using SSR Markers MSc. Thesis Messele Molla September, 2020 Addis Ababa, Ethiopia Genetic Diversity and Population Structure of Zymoseptoria tritici Populations of Southern Ethiopia Using SSR Markers MSc. Thesis Submitted to Institute of Biotechnology, Addis Ababa University, in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biotechnology By Messele Molla September, 2020 Addis Ababa, Ethiopia ii iii Acknowledgement First of all, I would like to thank the Almighty God for bringing me this far in my academic pursuit. I would like to thank my thesis advisor Dr. Teklehaimanot Hailesilassie. I am very grateful for his immensely helpful comments and suggestions to complete this thesis work. I would like to forward the deepest of my appreciation and gratitude to my advisor Dr. Kassahun Tesfaye for all supports. I would like to express my sincere gratitude to Tilahun Mekonnen for his continuous guidance, critical comments, patience, motivation, enthusiasm, and immense knowledge. I credit every piece of strength of this study to my advisor Tilahun Mekonnen and any weakness to myself. My deepest gratitude also goes to Obssi Dessalegn for his patience and constructive advice throughout the course of the thesis work. I would also like to extend my thanks to the Institute of Biotechnology of Addis Ababa University for the chance I got to attend the MSc. program in Biotechnology. My sincere gratitude also goes to Ethiopian Institute of Agricultural Research for granting the study. I am also very grateful to the National Agricultural Biotechnology Research Center, Holetta, for the laboratory space and facilities. Many thanks to Zewdinesh Damtew for her skillful assistance in molecular lab work. We spent fantastic time in the lab. Thanks a lot for the benefits I got from her as a result of our close familiarity. iv I would like to express my warm thanks and regards to my dear family Eleni Yalew, Molla Kassia, Fentaye Tareke, and others for their frenziedly love and hope for my pleasure and better life. Thank you very much for your love and support. v Table of Content Contets Page Acknowledgement .................................................................................................................... iv List of Figures ......................................................................................................................... viii List of Tables ............................................................................................................................ ix List of Appendices ......................................................................................................................x List of Abbreviations................................................................................................................. xi ABSTRACT: ........................................................................................................................... xii 1. INTRODUCTION ...............................................................................................................1 1.1 Objectives ..........................................................................................................................3 1.1.1 General objectives .......................................................................................................3 1.1.2 Specific objectives .......................................................................................................4 2. LITERATURE REVIEW ........................................................................................................5 2.1 Origin and Distribution of Zymoseptoria tritici ..................................................................5 2.2 Taxonomy and Biology of Zymoseptoria tritici ..................................................................6 2.3 Genetics of Zymoseptoria tritici .........................................................................................7 2.4 Zymoseptoria tritici Infection Process and Epidemiology ...................................................8 2.5 Septoria tritici Blotch Disease Symptom.......................................................................... 10 2.6 Impact of Septoria tritici Blotch Disease on Wheat Production ........................................ 11 2.7 Septoria tritici Blotch Control Methods ........................................................................... 12 2.7.1 Chemical control method ........................................................................................... 12 2.7.2 Cultural method ......................................................................................................... 13 2.7.3 Resistance cultivars ................................................................................................... 13 2.8 Genetic Diversity of Zymoseptoria tritici ......................................................................... 14 2.9 DNA markers used for Zymoseptoria tritici genetic analysis ............................................ 15 2.10 Measuring Molecular Variability of a Population ........................................................... 16 3. MATERIALS AND METHODS ........................................................................................... 19 3.1 Description of the Study Area .......................................................................................... 19 3.2 Sample Collection .......................................................................................................... 20 3.3 Diagnostic and Disease Assessment in the Field .............................................................. 21 3.4 Isolation of Septoria tritici Pathogen ................................................................................ 22 vi 3.5 Assessing the Genetic Diversity of Zymoseptoria tritici Isolates ...................................... 24 3.5.1 Genomic DNA isolation ............................................................................................ 24 3.5.2 Molecular detection of Zymoseptoria tritici ............................................................... 24 3.5.3 SSR marker based genotyping ................................................................................... 25 3. 5.4 Data scoring and statistical analysis .......................................................................... 26 4. RESULTS ............................................................................................................................. 28 4.1 Molecular Based Identification of Zymoseptoria tritici..................................................... 28 4.2. Molecular Diversity Analysis .......................................................................................... 29 4.2.1 Microsatellite markers level of polymorphism ........................................................... 29 4.2.2 Genetic variability within and among the populations ................................................ 33 4.2.3 Analysis of molecular variance (AMOVA) ................................................................ 34 4.2.4 Measures of Genetic identity and Genetic distance between the populations .............. 34 4.2.5 Cluster analysis ......................................................................................................... 35 4.2.6 Principal coordinate analysis ..................................................................................... 37 4.2.7 Population structure and admixture pattern ................................................................ 38 5. DISCUSSION ....................................................................................................................... 40 6. CONCLUSION ..................................................................................................................... 45 7. RECOMMENDATION ......................................................................................................... 46 8. REFERENCES ..................................................................................................................... 47 9. APPENDICES ...................................................................................................................... 58 vii List of Figures Figure 1: Global distribution of Septoria spp.. .............................................................................6 Figure 2: Septoria tritici blotch disease cycle. .......................................................................... 10 Figure 3: Symptoms of Septoria tritici blotch on wheat leaf ...................................................... 11 Figure 4: Map of Ethiopia (on the left) with the four zones (on the right) .. ............................... 20 Figure 5: Z.ymoseptoria tritici isolation procedure.. .................................................................. 23 Figure 6: PCR products for molecular diagnostics of Zymoseptoria tritici.. ............................... 28 Figure 7: Neighbor-joining tree for 51 isolates of Z. tritici based on Jaccard coefficient . .......... 36 Figure 8: Two dimensional plot of PCoA analysis of 51 Z. tritici isolates .................................. 37 Figure 9: Inferred population structure of 51 Z. tritici isolates . ................................................. 39 viii List of Tables Table 1 Description of areas from where samples of Zymoseptoria tritici